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Exosomes carry specific protein and nucleic acid cargo that can serve as biomarkers of many diseases including HIV, cancer, and neurodegenerative diseases4,5,6,7,8,9,10. Furthermore, the cargo-carrying capacity of exosomes is being leveraged to deliver therapeutic agents5. Exosomes are 30–150 nm extracellular vesicles (EVs) secreted into the extracellular milieu by most cell types. They have been detected in body fluids such as blood, urine, and semen, as well as extracellular matrix in tissues, and carry cargo involved in mediating communication between cells and organs. Exosome protein and nucleic acid cargo is influenced by cell type of origin, and biological processes including immune activation, inflammation, and exposure to stressors6,11,12,13. Exosomes have physiological functions that contribute to maintaining human health, but have also been implicated in contributing to pathophysiology of many diseases5,13,14,15,16.

HIV and other retroviruses exploit pre-existing cellular pathways for vesicle trafficking, assembly, and release for viral dissemination and pathogenesis9,10,17,18,19,20. Furthermore, HIV infection induces exosome release from monocytes21, macrophages20, dendritic cells22, and T cells16. Exosomes secreted from HIV-infected cells transport viral and host components that can facilitate viral dissemination through body fluids19,22,23,24,25,26,27. Most studies on exosomes in HIV infection have focused on detrimental effects, including pro-inflammatory or pro-apoptotic effects8,16,18,22,23,25,26. For example, HIV Nef protein was shown to induce release of Nef-positive exosomes from T cells, leading to bystander apoptosis in vitro16. However, exosomes can also play beneficial roles. For example, exosomes can promote antiviral activity by cell-to-cell transfer of innate antiviral factors such as APOBEC3G, and cytokines that inhibit viral replication such as type I interferons28,29. Exosomes may also serve protective functions during oxidative stress30.

A previous study showed that HIV-infected individuals have more abundant plasma exosomes compared to healthy controls7. HIV infection also influences the size and cargo of other plasma EVs7,8. However, little is known about the relationship of circulating exosome cargo to immune responses, and how these processes are altered by HIV infection. Here, we perform a cross-sectional study to characterize the protein cargo of circulating exosomes in a cohort of HIV-positive subjects on ART and uninfected HIV-negative controls, and determine the relationship of exosome protein cargo to virological, immunological, and oxidative stress markers. We then examine transcriptional changes induced in recipient monocytic cells following uptake of patient-derived exosomes to evaluate their functional effects on immune activation and inflammation.

To confirm proteins of interest detected by proteomics using another method, we performed immunoblotting to probe 3 proteins that could potentially play a functional role in immune activation signaling (ADAM10, DDR1, Notch4) in exosome samples. Among these candidates, only Notch4 was detected by immunoblotting of these plasma exosome fractions (Fig. 4, top left). Therefore, further studies focused on Notch4. Notch4 levels in plasma exosomes from HIV-viremic subjects were higher than those in HIV-aviremic and HIV-negative subjects based on ELISA (Fig. 4, top middle panel) and immunoblotting (data not shown). Although Notch4 levels increased with increasing EV numbers, the correlation did not reach statistical significance (p = 0.07, Supplementary Figure S6). K:T ratio was increased in both aviremic and viremic HIV-positive subjects (Fig. 4, top right panel). Plasma exosome-associated Notch4 correlated positively with K:T ratio and exosome-associated-HLA-DRA measured by ELISA (Fig. 4, bottom middle and right panels) and negatively with CD4/CD8 ratio, an indicator of poor CD4 T cell recovery associated with increased immune activation (Fig. 4, bottom left). Thus, Notch4 is a novel plasma exosome-associated protein identified by untargeted proteomics that correlates with immune markers and exosomal HLA-DRA.

Notch4 is expressed in human dendritic cells

Next, we investigated potential sources of plasma exosomes carrying Notch4 protein. Notch4 mRNA is highly expressed in human dendritic cells and natural killer (NK) cells based on gene expression profiles of different immune cell populations from the Immgen Consortium Database (www.immgen.org), suggesting these immune cells are a potential source of exosomal Notch4 (Fig. 5, top panel). To further investigate this possibility, we used flow cytometry with antibodies against Notch4 and immune cell markers to assess staining on freshly isolated PBMCs from 3 randomly selected healthy donors. Notch4 staining was detected in a minority of plasmacytoid and myeloid dendritic cells (Fig. 5, middle and bottom panels), and although it can also be expressed on monocytes, NK cells, or lymphocytes it is generally at much lower levels (data not shown). Thus, circulating dendritic cells are one potential source of exosomes carrying Notch4. To confirm this, we generated monocyte-derived dendritic cells (MDDC) in vitro from 3 randomly selected healthy donors, induced maturation with LPS treatment (Fig. 6, top left)45, and isolated exosomes from MDDC conditioned medium before and after LPS treatment to activate MDDCs and mimic immune activation conditions2. NTA analysis showed an increase in EV concentrations after LPS treatment in all 3 donor samples (Fig. 6, bottom panels). Immunoblotting was performed on mature MDDC-whole cell lysates (WCL) and exosome fractions for exosome markers, Notch4, and GAPDH (Fig. 6, top right). Exosome markers (CD9 and CD81) and Notch4 levels in exosome fractions, but not WCL fractions, increased with increasing EV numbers based on western blot band intensities. These results suggest that dendritic cells are a potential source of Notch4 in circulating plasma exosomes.

Discussion

In this study, we show that HIV-positive subjects on ART have higher abundance of circulating plasma exosomes as compared to uninfected controls matched for demographics and smoking. The exosome markers CD9, CD63, and HSP70 were elevated in plasma from HIV-positive subjects on ART; CD9 and CD63 were elevated even in aviremic HIV-positive subjects. These findings contrast to a previous study by Hubert et al.7, which showed that circulating plasma exosomes are more abundant (as measured by an indirect assay using acetylcholinesterase activity) in ART-naive, but not ART-suppressed subjects, relative to healthy controls. Contrary to expectations, EV numbers or exosome markers such as CD9 and CD63 did not correlate with plasma VL, CD4 counts, and CD4:CD8 ratio in HIV-positive subjects on ART. This finding is consistent with Hubert et al.7, which reported that exosome abundance correlated with CD4 counts in ART-naive HIV-positive subjects with high VL (mean 10,000 HIV RNA copies/ml), but not in ART-suppressed subjects. Our findings suggest that circulating exosomes are increased in patients with treated HIV infection, but the levels do not show a clear relationship to virological and immunological parameters in treated patients with suppressed VL.

Several studies reported effects of HIV infection on exosome secretion, abundance, and cargo in vitro22,25,51. Although some studies suggested that circulating exosome cargo may be related to chronic inflammation in HIV infection7,8, few studies have investigated circulating exosomes as biomarkers. By untargeted proteomics of exosomes immunoaffinity-purified from plasma of HIV-positive subjects on ART and healthy controls, we identified exosome protein cargo related to myeloid cells, immune activation, inflammation, and oxidative stress. Of 321 exosome-associated proteins detected by proteomics, at least 40 were related to immune activation/inflammation, of which 9 were detected only in HIV-positive exosomes including CD14, CRP, HLA-A, HLA-B, and ITGB1. CRP is an inflammation marker secreted by the liver in response to IL-6 stimulation52. To our knowledge, this is the first study detecting CRP in exosomes, although monomeric CRP has been detected in circulating microparticles in the setting of myocardial infarction53. CRP levels are higher in individuals with HIV infection, often rising over time54 in association with HIV disease progression55. We identified CD14 in plasma exosomes, suggesting that myeloid cells are a likely source of circulating exosomes; however, platelets are another potential source based on our detection of platelet-associated markers in exosome fractions. Another category of proteins detected in exosomes were proteins related to oxidative stress including CAT, ENO1, PRDX1, PRDX2, PXDN, SEPP1, and TXN. These proteins are involved in reducing oxidative stress, and were detected more frequently in HIV-negative exosomes.

We show here, for the first time, the presence of Notch4 in plasma exosomes. Notch4 is a receptor for Jagged-1, Jagged-2, and Delta-1, and regulates cell-fate determination. The intracellular domain is activated by proteolytic cleavage and translocates to the nucleus, where it forms a transcriptional activator complex. While Notch1, 2 and 3 have been previously detected in exosomes56,57,58,59, our study is the first to provide evidence for Notch4 in plasma exosomes by LC-MS/MS and immunoblotting. Furthermore, we provide evidence that myeloid and plasmacytoid dendritic cells are one potential source of Notch4-containing exosomes in plasma based on gene expression and flow cytometry, which detected Notch4 expression in myeloid and plasmacytoid dendritic cells, and correlation between exosomal Notch4 and HLA-DRA levels. Notch signaling is critical for differentiation, development, and functions of dendritic cells, including plasmacytoid dendritic cells responding to viral stimulation by IFN-α secretion60,61,62. Dendritic cell secretion of cytokines/chemokines and downstream polarization of Th-type responses are also regulated by Notch signaling63. Notch4 is expressed by endothelial cells64, which represent another potential source of Notch4-containing exosomes. However, we assayed exosome fractions for endothelial marker CD105 by ELISA and found no correlation with exosomal Notch4 levels (data not shown); examination of other potential sources requires further study. Exosomal Notch4 correlated with increased K:T ratio, decreased CD4/CD8 ratio, and exosomal HLA-DRA, which are indicators of immune activation in HIV infection41,65,66. Furthermore, Notch4 was detected in exosomes released from mature MDDCs following treatment with LPS in vitro. These findings suggest that exosomal Notch4 is a potential biomarker of immune activation in HIV infection.

Previous studies show immunomodulatory effects in monocytic cells induced by exosomes14. We therefore hypothesized that plasma exosomes of HIV-positive individuals on ART may have immunomodulatory effects on recipient cells. We tested this hypothesis using a cellular model in which THP-1 monocytes were incubated with IAP-purified plasma exosomes from HIV and control subjects; changes in gene expression were examined using the NanoString platform, which utilizes digital color-coded barcodes to detect and count hundreds of unique transcripts in a single reaction, without PCR amplification67. Genes associated with interferon responses, innate immune responses, and inflammation, were upregulated in THP-1 cells following incubation with exosomes; many genes induced by exosome treatment were also induced by IFN-γ and/or LPS. These findings suggest that circulating exosomes can have pro-inflammatory and other immunomodulatory effects on recipient cells. However, these effects were not specific to plasma exosomes from HIV-positive subjects: in 2 of 4 healthy control donors, plasma exosomes induced gene expression changes similar to those induced by HIV-positive exosomes. Triggering of cell signaling and gene expression changes are not unique to exosomes from diseased/stressed conditions14,68. We cannot exclude the possibility that modulation of some analyzed genes was the result of secondary events subsequent to exosome stimulation of THP-1 cells. Nonetheless, we found that HIV-positive exosomes had stronger effects on recipient cells compared to HIV-negative exosomes.

We acknowledge limitations of the study, particularly those related to the purity of exosome preparations69. Although plasma is a good source of exosomes, it is challenging to separate plasma exosomes from abundant plasma proteins, larger microvesicles, subcellular fractions, and cholesterol particles. Our study was also limited by small volumes of plasma available for exosome isolation. Exosome isolation by differential ultracentrifugation yielded exosome fractions of higher purity than the Exoquick method, but required larger starting volumes (>1 ml) and pooling of multiple samples. Importantly, both methods gave similar results with regard to our main finding that exosome markers are increased in HIV-positive compared to HIV-negative plasma. The optimal method to isolate exosomes from small volumes of plasma is to precipitate exosomes using an exosome-precipitating reagent to minimize exosome loss, and then purify the exosome fraction by another method such as immunoaffinity purification. For proteomics, we purified plasma exosome fractions by depleting 12 abundant plasma proteins, followed by IAP using exosome marker antibodies. This method resulted in higher purity of exosome fractions, but lower exosome yield. Another limitation of our study was lack of availability of primary DCs from HIV-infected patients, which would be more relevant to study sources of Notch4-bearing exosomes during HIV infection. Likewise, future studies using primary monocytes would be helpful to validate our findings from THP-1 cells. Additional limitations of our study include the relatively small sample size and high rates of comorbidities such as smoking, HCV-coinfection, and cocaine or alcohol abuse, which could influence some findings. High rates of smoking and cocaine use in our study cohort may have influenced markers of inflammation and oxidative stress70,71,72,73,74, but we did not have sufficient statistical power to evaluate effects of these covariates. Further studies are needed to address the impact of these and other lifestyle factors on exosomes.

In conclusion, our study shows that: (1) HIV-positive individuals have elevated abundance of plasma exosomes and exosome markers (CD9, CD63 and HSP70), and these changes correlate with metabolites indicative of oxidative stress and immune activation; (2) Notch4 protein is detected in plasma exosomes, and correlates with immune activation markers in HIV-positive subjects; (3) plasmacytoid and myeloid dendritic cells express Notch4 mRNA and protein, and are one potential source of exosomes in plasma of HIV-positive subjects; and (4) circulating exosomes in ART-treated HIV-positive subjects carry protein cargo related to immune activation and oxidative stress, have immunomodulatory effects on myeloid cells, and may have pro-inflammatory and redox effects during HIV pathogenesis. Understanding the relationship of exosome cargo to HIV pathogenesis, immune activation, and oxidative stress may contribute to novel biomarker discovery, elucidate mechanisms contributing to disease progression and comorbidities, and accelerate the identification of new biomarkers and therapeutics.

Methods

Study subjects

The study was performed in accordance with guidelines in the Declaration of Helsinki. Plasma samples from HIV-positive subjects (n = 43, age 35–65 years) were from the National NeuroAIDS Tissue Consortium (NNTC) [Manhattan HIV Brain Bank (n = 8), National Neurological AIDS Bank (n = 4), California NeuroAIDS Tissue Network (n = 12), Texas NeuroAIDS Research Center (n = 3)] and AIDS Linked to the Intravenous Experience (ALIVE) cohort (n = 16). All subjects were enrolled with written informed consent and IRB approval at each study site (IRB committees at Mount Sinai School of Medicine and Mount Sinai Hospital, UCLA, University of California San Diego, and Johns Hopkins Bloomberg School of Public Health, respectively). Inclusion criteria for HIV-positive subjects were: combination ART use with HIV plasma VL undetectable or below 2500 HIV RNA copies/ml. For NTA measurements only, we included 4 HIV-positive subjects not reporting ART use at time of plasma collection (plasma VL range, 507 to 2,405 HIV RNA copies/ml). Exclusion criteria were diagnoses or lab values indicative of renal or liver failure. Healthy control plasma samples (n = 34), were from HIV- negative donors (from Bioreclamation IVT, n = 29; ALIVE cohort, n = 5) with informed consent and IRB approval from Dana-Farber Cancer Institute. The healthy control group was frequency-matched to the HIV-positive group by age, race, gender, and smoking (Table 1).

Differential Ultracentrifugation method

Exosome fractions were isolated by differential ultracentrifugation as described76. Plasma samples were pooled from HIV-positive and HIV-negative subjects (n = 15 each, see Supplementary Figure S1). For each sample, 1.3 ml plasma was centrifuged at 500 × g and the supernatant was collected and diluted 1:2 with PBS. Diluted plasma was centrifuged at 12,000 × g for 30 minutes and the supernatant was passed through 0.22 μm filter to remove EVs larger than exosomes. The filtrate was further diluted with PBS such that plasma was finally diluted 1:5 in PBS. Diluted plasma was ultracentrifuged at 120,000 × g for 70 minutes at 4 °C. Supernatant was decanted and exosome pellet resuspended in 100 μL PBS.

Metabolomic profiling

Untargeted metabolomic profiling was performed by Metabolon (Durham, NC) combining three independent platforms: ultra-high performance liquid chromatography and tandem mass spectrometry (UHLC/MS2/MS) optimized for detection of acidic metabolites, UHLC/MS2/MS optimized for detection of basic metabolites, and gas chromatography (GC)/MS. Plasma samples (100 ul) were extracted using the MicroLab STAR system as described78. Briefly, protein was precipitated from plasma with methanol containing four standards to monitor extraction efficiency. The resulting supernatant was split into equal aliquots for analysis on the three platforms. Aliquots, dried under nitrogen, were subsequently reconstituted in 50 μL 0.1% formic acid in water (acidic conditions) or in 50 μL 6.5 mM ammonium bicarbonate in water, pH 8 (basic conditions) for the two UHLC/MS/MS analyses or derivatized to a final volume of 50 μL for GC/MS analysis using equal parts bistrimethyl-silyl-trifluoroacetamide and solvent mixture acetonitrile:dichloromethane:cyclohexane (5:4:1) with 5% triethylamine at 60 °C for one hour. Three types of controls were utilized: samples derived from pooled experimental samples served as technical replicates, extracted water samples served as blanks, and a cocktail of standards spiked into every analyzed sample allowed instrument performance monitoring. The UHLC/MS2/MS platform was based on a Waters ACQUITY UHPLC and Thermo-Finnigan LTQ mass spectrometer, which consisted of an electrospray ionization source and linear ion-trap mass analyzer. Derivatized samples for GC/MS were separated on 5% phenyldimethyl silicone columns, with helium as the carrier gas and a temperature ramp from 60 °C to 340 °C over a 16-minute period. Analysis was performed on a Thermo-Finnigan Trace DSQ fast-scanning single-quadrupole mass spectrometer operated at unit mass resolving power with electron impact ionization and a 50–750 atomic mass unit scan range. Compounds were identified by automated comparison of the ion features in the experimental samples to a reference library of over 4, 000 chemical standard entries that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments as well as associated MS spectra and curated by visual inspection for quality control using software developed at Metabolon79.

Proteomic analysis

To obtain purified exosome fractions for proteomic analysis, 12 abundant plasma proteins were immunodepleted from plasma using Proteome Purify™ 12 kit (R&D systems) and albumin was depleted with 2 rounds of albumin depletion using AlbuSorb™ - Albumin Depletion Kit (Biotech Support Group). Plasma was pre-cleared using Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology) and exosome fraction precipitated using ExoQuick. Exosome fractions were further purified by immunoprecipitation from plasma exosome fractions using antibodies against CD9, CD63, and CD81 (#EXOFLOW32A-CD81, −CD63, −CD9, System Biosciences and # EX-COM-SP, JSRmicro). Isolated exosomes were analyzed by mass spectrometry using two different platforms: (1) ABSciex 4800Plus MALDI-TOF/TOF mass spectrometer and (2) Thermo Scientific LTQ-Orbitrap ion-trap mass spectrometer. In experiments using the first platform, samples were processed as follows: following immunoprecipitation, exosome-conjugated beads were washed thrice and exosomes were eluted as described80 using low pH buffer (50 mM glycine, pH 3.0) followed by neutralization of pH with 1 M Tris-HCl (pH 8.0). To ensure that exosomes were disrupted, detergent extraction was employed using deoxycholate. Protein was precipitated using cold methanol, then reduced, alkylated, and digested overnight using trypsin. Samples were run on nanoflow LC in reverse phase on a 15 cm C18 PepMap column on an LC Packings/Dionex nanoflow LC, and mass spectrometry was done on an ABSciex 4800Plus MALDI-TOF/TOF mass spectrometer. For peptide mapping and protein identification, database searches were performed using ProteinPilot 4.5b (ABSciex, Framingham, MA). Protein identifications with at least 95% confidence as determined by ProteinPilot were considered significant. In experiments using the second mass spectrometry platform, samples were processed as follows: following immunoprecipitation, exosome-conjugated beads were washed thrice and exosomes were solubilized with 0.5% RapiGest and boiling at 100 °C for 5 min. Residual immunoglobulins were depleted with Protein A/G PLUS-Agarose beads. Proteins were then digested overnight using trypsin (Sequence grade; Promega). Samples were then dried in a speed-vac and rehydrated in 50 µl of 0.5% TFA. The sample was then run through a stage tip (C18 tip), washed, and eluted. The solution was dried and then rehydrated in 2.5% acetonitrile and 0.1% formic acid solution. Reverse-phase fractionation was done on a 25 cm C18 column and samples were run on LTQ-Orbitrap ion-trap mass spectrometer (ThermoFisher). Database search was performed using the software program, Sequest (ThermoFisher). Common contaminants (keratin, bovine proteins, mouse/rabbit IgGs) were omitted from downstream analysis. Remaining proteins identified were compared against existing exosome database (www.exocarta.org) and the top 200 most abundant plasma proteins published previously81. Functional annotation was performed by GO mapping using PANTHER (pantherdb.org) and Biobase (genexplain.com/transfac).

MDDC culture and exosome isolation

PBMCs were isolated from blood samples of 3 healthy donors using Histopaque-1077 (Sigma). Fresh PBMCs were seeded (6 × 106 cells/ml) in 30 ml RPMI 1640 media supplemented with 10% FBS (depleted of exosomes by ultracentrifugation at 100,000 × g) and 1% Penicillin-Streptomycin. Following monocyte enrichment by plastic adherence (8–10% cells attached), differentiation to MDDC was induced by GM-CSF (800 U/ml) and IL-4 (500 U/ml) treatment82. After 5 days in culture, MDDCs were treated with LPS (100 ng/ml) for an additional 48 hrs to induce maturation45. Conditioned media was collected from mature MDDC, and centrifuged at 300 × g for 5 min and 3000 x g for 15 min. The supernatants were passed through 0.22 μm filter, and exosomes were then isolated using Exoquick TC (System Bioscience) per manufacturer’s instructions. The exosome pellet was re-suspended in 40 µl PBS.

Functional analysis of plasma exosomes in THP-1 recipient cells

THP-1 suspension cells were cultured in 6-well plates at 2.5 × 105 cells/ml in RPMI 1640 media supplemented with 1 mM sodium pyruvate, 10% FBS (depleted of exosomes by ultracentrifugation at 100,000 × g) and 1% penicillin-streptomycin. Cells were treated for 72 hrs with IAP-purified plasma exosomes (20 μg) from HIV-positive (n = 4) and healthy controls (n = 4). THP-1 cells treated with PBS and “shaved exosomes” pre-treated with Proteinase-K (to shave off surface proteins) were used as negative controls83. Proteinase K was inactivated by incubation at 70 °C for 15 min. Prior to exposing THP-1 cells to “shaved exosomes”, cells were treated with Proteinase K for 30 minutes and washed twice with PBS to remove excess Proteinase K. THP-1 cells were treated with the exosomes for 72 hrs. THP-1 cells treated with IFN-gamma (100 IU/ml) or LPS (0.5 μg/ml) for 24 hrs served as positive controls. Treated cells were collected and rinsed in PBS. Total RNA was isolated using the MirVana kit (Thermo Fisher Scientific). RNA content and quality was evaluated by BioAnalyzer (Agilent). mRNA hybridization, detection, and scanning were performed on 100 ng of total RNA using NanoString Counter technology84 with probes for 770 genes in the PanCancer Immune Profiling Panel and 30 PLUS custom probes (NanoString Technologies, Seattle, WA) at the DFCI Molecular Biology core facility.

Data processing, bioinformatics, and statistical analysis

For metabolite profiling, metabolite data was normalized by median centering. Missing values were imputed with the lower limit of detection for a given metabolite. Batch normalization was performed using the median ratio for each metabolite in duplicate “anchor” samples across runs. Significantly altered metabolites were defined by FC > 1.3, p-value < 0.05, and FDR < 0.10. Statistical analyses using Welch’s t-test (p < 0.05) were performed on log-transformed data. Pearson correlations were used to evaluate relationships between plasma metabolites and exosome marker levels (p < 0.05). Metabolite clusters were identified by unsupervised hierarchical clustering using the heatmap.2 function of R. For Nanostring gene expression profiling, raw counts were normalized after quality control-check, and fold-change calculations and heatmaps were constructed using nSolver 3.0 software. Significantly altered genes were defined by FC > 1.3, p-value < 0.05 and FDR < 0.10. False-discovery rates for metabolite and gene expression profiling were estimated using fdrtool in R. Exosome marker levels were compared between groups using the Mann Whitney U-test in PRISM (p < 0.05).

Data availability

All data generated or analyzed during this study are included in this published article (and its Supplementary Information files) or available from the corresponding author on reasonable request.

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Contributions

S.C. performed experiments, organized inventories, analyzed data, drafted the manuscript, and prepared figures and tables. D.L. performed bioinformatics and statistical analysis, V.M. organized clinical data, performed data analysis, and prepared figures. S.D. performed proteomic analysis; R.K.R. and C.M. participated in flow cytometry studies and data analysis. S.M., G.K. and S.H.M. provided clinical samples and data, and participated in cohort selection and data analysis. D.G. designed and supervised the study and participated in data analysis and drafting the manuscript. All authors read, participated in editing the manuscript, and approved the final manuscript.

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